Two types of factors found during the search of growth factors for fibroblast cells of normal rats as molecules enhancing the transformation of normal cells were given names as transforming growth factor-α (TGF-α) and transforming growth factor-β (TGF-β). Subsequent studies have revealed that TGF-α is a molecule belonging to the epidermal growth factor (EGF) family, whereas TGF-β is produced from almost all types of cells and further the receptor therefor is expressed in a wide variety of organs and cells (Biol. Signals., Vol. 5, p. 232, 1996 and Pulmonary Fibrosis, Vol. 80 of Lung Biology in Health and Disease Series, ed. by Phan et al, p. 627, Dekker, New York, 1995).
TGF-β has the activity to regulate cell differentiation and growth. The cell growth-promoting activity of TGF-β largely depends on the type of cell (Roberts et al, The transforming growth factor-βs, In Peptide Growth Factors and Their Receptors, Part I, ed. by Sporn, M. B. & Roberts, A. B. Springer-Verlag, Berlin, 1990, p. 419-472). For example, it has been clarified that the factor shows cell growth-promoting activity to mesangial cells, such as fibroblast cells and vascular smooth muscle cells, whereas it serves not as a growth-promoting factor but as a growth-suppressing factor on a variety of cells including epithelial cells, vascular endothelial cells, and hemocytes. TGF-β has been further revealed to possess not only cell growth modulating functions but also various functions including immune system regulation; enhancement of the extracellular matrix (ECM) protein accumulation, such as collagen, fibronectin, and tenascin (Adv. Immunol., Vol. 55, p. 181, 1994 and Seminars in Cell Biol., Vol. 5, p. 389, 1994); and so on.
Recently, it has been clarified that the mechanism for the diversified functions of TGF-β depends on the distinctive structure of TGF-β receptors and the signal transduction thereby.
TGF-β is a protein with a molecular weight of about 25-kDa. Two peptide strands form a dimer. Five types of isoforms exist for TGF-β: i.e., TGF-β1, TGF-β2, and TGF-β3 in mammals; TGF-β4 in chicken; and TGF-β5 in frog. These isoforms exhibit a homology of about 70% to one another.
Among these TGF-β species, the function of TGF-β1 has been extensively analyzed. TGF-β1 plays extremely important roles in the process of wound healing in biological tissues (New Engl. J. Med., Vol. 331, p. 1286, 1994 and J. Cell. Biol., Vol. 119, p. 1017,1992). In the site of tissue wounded, rapid and dynamic biological reactions including infiltration of inflammatory cells and fibroblast cells, production of ECM and vascularization, and cell growth for the subsequent tissue regeneration occur to repair the injured tissue.
First, bleeding starts at the infliction of a wound, and then, TGF-β and PDGF (platelet-derived growth factor) are produced by platelets together with the activation of ECM-bound inactive TGF-β at the wound site. By exposure to a high concentration of thus produced TGF-β, cells migrated to the wound site and cells at the wound site secrete growth factors and cytokines, such as FGF (fibroblast growth factor), TNF (tumor necrosis factor), and IL-1 (interleukin-1); and fibroblast cells also synthesize and secrete ECM.
Further, for example, increased production of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF; also celled Hcs24) (J. Cell Biology, Vol. 114, No. 6, p. 1285-1294, 1991; Int. J. Biochem. Cell Biol. Vol. 29, No. 1, p. 153-161, 1997; Circulation, Vol. 95, No. 4, p. 831-839, 1997; Cell Growth Differ., Vol. 7, No. 4, p. 469-480, 1996; J. Invest. Dermatol., Vol. 106, No. 4, p. 729-733, 1996; J. Invest. Dermatol., Vol. 105, No. 2, p. 280-284, 1995; J. Invest. Dermatol., Vol. 105, No. 1, p. 128-132, 1995; and international publication WO 96/38172)), and fibronectin are observed in fibroblast cells and mesangial cells.
Further, TGF-β1 is believed to contribute to the wound healing by suppressing the production of proteases and enhancing the production of inhibitors against the enzymes, and further, by enhancing the synthesis of integrins, that participate in the adhesion of ECM to cells, promoting the production and deposition of ECM. In addition, TGF-β also exhibits an immuno-suppressing activity by suppressing the functions of T lymphocytes and B lymphocytes to inhibit the synthesis of TNF and IL-1.
The mechanism for the regulation of TGF-β expression has not been fully clarified, but the expression is expected to be regulated by the binding of TGF-β itself to proteoglycan, i.e. ECM (Nature, Vol. 346, p. 281, 1990 and Nature, Vol. 360, p. 361, 1992). More specifically, it has been believed that, the overexpression of TGF-β is suppressed by a negative regulation by ECM, whose production is enhanced by TGF-β itself, while TGF-β promotes the wound healing. Therefore, abnormalities in the negative regulation may lead to the overexpression of TGF-β, and thus can result in a morbid state, such as tissue fibrosing (fibrosis).
On the other hand, in pulmonary fibrosis and nephrosclerosis, in spite of sufficient deposit of ECM the concentration of TGF-β is retained high and leads to the progress of the morbid states, such as fibrosis (Kidney Int. Vol. 45, p. 916, 1994 and J. Clin. Invest., Vol. 92, p. 632, 1993). The unceasing infliction of tissue injury has been presumed to continuously transduce signals to express TGF-β, suppress the above-mentioned negative regulation signal for TGF-β expression, or cause both events synergistically in pulmonary fibrosis and nephrosclerosis.
Nephrosclerosis is a terminal state of many types of kidney diseases, such as chronic glomerulonephritis and diabetic nephropathy, which is characterized by the proliferation of mesangial cells and the production of ECM. Aberrant expression patterns of TGF-β have been found in kidney of nephrosclerosis patients (J. Clin. Invest., Vol. 90, p. 1, 1992 and Proc. Natl. Acad. Sci. USA, Vol. 86, p. 1056, 1989). Further, in an experimental model for nephritis, which is induced by an anti-Thy-1 antibody, the administration of anti-TGF-β antibody was shown to suppress the progress of nephritis. This suggests that TGF-β participates in the onset of morbid state of nephrosclerosis (Nature, Vol. 346, p. 371, 1990).
On the other hand, TGF-β is expressed at high concentration in lung of subjects with pulmonary fibrosis induced by bleomycin administration or sudden pulmonary fibrosis; suggesting the relation of TGF-α in the onset of pulmonary fibrosis.
Further, expression of TGF-β was detected at collagen-deposited sites in tissues biopsy obtained from chronic hepatitis patients and cirrhosis patients. Furthermore, ECM deposition and TGF-β expression are also reported in vascular restenosis; arthritis, such as rheumatoid arthritis; keloid of skin; and so on.
These findings have suggested the possibility that TGF-β is associated with the onsets of morbid states of various types of tissue fibrosing (fibrosis). Thus, experimental attempts of therapy for tissue fibrosis are made by suppressing the function of TGF-β using antisense pharmaceuticals and gene therapy (Kidney Int., Vol. 50, p. 148, 1996).
Signal transduction into cells through the binding of TGF-β to TGF-β receptor initiates the above-mentioned expression of various functions of TGF-β and the onsets of various morbid states of tissue fibroses due to TGF-β.
Three types of TGF-β receptors have been identified from mammals including human and rat, which structures have been already revealed. The three are: the type I receptor (molecular weight=about 53 kDa; GenBank Accession No: L11695; Cell, Vol. 75, No. 4, p. 681, 1993; hereinafter referred to as “TGF-β type I receptor” or “TβRII”); the type II receptor (molecular weight=about 70 kDa; GenBank Accession No: M85079; Cell, Vol. 68, No. 4, p. 775, 1992; hereinafter referred to as “TGF-β type II receptor” or “TβRI”); and the type III receptor (molecular weight=about 200 to 300 kDa; GenBank Accession No: L07594; Cell, Vol. 67, No. 4, p. 785, 1991; Cell, Vol. 67, No. 4, p. 797, 1991; and Biochem. Biophys. Res. Commun. Vol. 189, No. 1, p. 356, 1992; hereinafter referred to as “TGF-β type III receptor” or “TβRIII”) (Adv. Imm., Vol. 55, p. 181, 1994).
The roles of the receptors have been revealed by functional analysis using artificially established TGF-β-resistant variant, derived from mink lung epithelium cell line MvlLu. Among the three receptors, TGF-β type I receptor and TGF-β type II receptor were demonstrated to be important and essential for the signal transduction of TGF-β (J. Biol. Chem., Vol. 265, p. 18518, 1990 and J. Biol. Chem., Vol. 266, p. 9108, 1991). On the other hand, TGF-β type III receptor is not essential for the signal transduction of TGF-β, and plays an indirect role in the transduction.
TGF-β type III receptor is a transmembrane protein consisting of 849 amino acids, which includes an extracellular domain (761 amino acids), transmembrane domain (24 amino acids) and cytoplasmic domain (43 amino acids). The cytoplasmic domain of the TGF-β type III receptor is rich in serine (Ser)/threonine (Thr) residues, which correspond to 40% or more of the total amino acids.
TGF-β type I receptor is a transmembrane protein consisting of 503 amino acids, which includes an extracellular domain (101 amino acids), transmembrane domain (22 amino acids) and cytoplasmic domain (356 amino acids).
Similarly, TGF-β type II receptor is a transmembrane protein consisting of 567 amino acids, which includes a signal sequence (23 amino acids), extracellular domain (136 amino acids), transmembrane domain (30 amino acids), and cytoplasmic domain (378 amino acids).
TGF-β type I and TGF-β type II receptors are single-transmembrane proteins whose extracellular domains are relatively short and which have a serine/threonine kinase structure containing two kinase domains. Further, the cytoplasmic domain contains an aspartic acid (Asp)-linked sugar chain, and the domain of the TGF-β type I receptor and TGF-β type II receptor have 10 cysteine (Cys) residues and 12 Cys residues, respectively, which provide a characteristic protein tertiary structure.
A region rich in glycine (Gly), serine (Ser), and threonine (Thr), dubbed “Gdomain”, is observed adjacent to the kinase domain in the cytoplasmic region of a TGF-β type I receptor. The TGF-β type II receptor has a similar structure to that of TGF-β type I receptor but lacks the GS domain.
The kinase domains of TGF-β type I receptor and TGF-β type II receptor have been demonstrated to share a homology of about 43%, and are both protein kinases specific to serine/threonine. Further, reaction of the two serine/threonine kinase domains have been revealed essential for the TGF-β signal transduction into cells (J. Biol. Chem., Vol. 269, p. 30753, 1994).
Both of TGF-β type I receptor and TGF-β type II receptor are essential for the signal transduction of TGF-β into cells. The TGF-β type II receptor can bind to TGF-β by itself, whereas the TGF-β type I receptor cannot bind to TGF-β alone. The formation of a complex of the TGF-β type I and type II receptors on the cell surface is important for the signal transduction of TGF-β into cells.
The binding of TGF-β to a TGF-β type II receptor has been reported to lead to the formation of a hetero-tetramer complex of the TGF-β type II receptor and TGF-β type I receptor on cell surface (J. Biol. Chem., Vol. 269, p. 20172, 1994). Specifically, when the TGF-β type I receptor and the TGF-β type II receptor form a complex in the presence of TGF-β, the TGF-β type I receptor serves as a substrate forTGF-β type II receptor and the GS domain thereof is phosphorylated by the TGF-β type II receptor to activate the TGF-β type I receptor. As a result, other intracellular substrates are phosphorylated and allows a further downstream transduction of the TGF-β signaling (Nature, Vol. 370, p. 341, 1994).
The TGF-β signaling pathway downstream of TGF-β type I receptor has not yet been fully elucidated, but recently, signaling molecules located downstream of the TGF-β type I receptor have been identified: (1) signaling molecules consisting of eight isoforms, collectively called “Smad” (S Mothers against Dpp), mediating the signal transduction of TGF-β (Nature, Vol. 381, p. 620, 1996; Cell, Vol. 86, p. 543, 1996; Nature, 383, p. 168, 1996; and Nature, Vol. 383, p. 832, 1996); and (2) a signaling molecule called TAK1 (TGF-β activated kinase-1) (Science, Vol. 270, p. 2008, 1995). Smad2 and Smad3 have been reported that they bind to the TGF-β type I receptor, which had been activated by forming a complex with the TGF-β type II receptor; are phosphorylated by the kinase domain of the TGF-β type II receptor; are released from the TGF-β type II receptor to form a complex with Smad4 (DPC4); and, then, translocate into the nucleus (Cell, Vol. 87, p. 1215, 1996 and EMBO J., Vol. 16, No. 17, 1997). The Smad2/Smad4 or Smad3/Smad4 complex translocated into the nucleus are suggested to function as a transcription activating factor by binding to a DNA-binding protein (transcription factor), so that gene expression is regulated (Nature, Vol. 383, p. 832, 1996 and Nature, Vol. 390, p. 465, 1997).
TAK1 has been demonstrated to be associated with the signal transduction of TGF-β, functioning as a MAPKKK in the cascade of MAP kinase.
As described above, TGF-β is closely involved in the onsets of nephrosclerosis; various tissue fibroses, such as pulmonary fibrosis and cirrhosis; as well as the onset of various morbid states, such as chronic hepatitis, rheumatoid arthritis, vascular restenosis, and keloid of skin. The onsets of these morbid states may result from TGF-β signal transduction into cells mediated by TGF-β receptor.
Accordingly, this raises the possibility that the morbid states can be treated or prevented by regulating, in particular suppressing, the transduction of TGF-β signal into cells.
An attempt to suppress the morbid states by suppressing TGF-β signal transduction is in practice to treat nephritis, and such, by suppressing the function of TGF-β using antibodies against TGF-β or antisense nucleic acids of the TGF-β gene, in which the TGF-β is a target.
However, treatment of diseases by suppressing functions of TGF-α receptors, particularly TGF-β type II receptor, which is the binding partner of TGF-β, hasn't been reported; in other words, there is no report on therapy for the diseases by inhibiting the signal transduction of TGF-β into cells mediated by the TGF-β type II receptor, in which the TGF-β type II receptor is a target.
Further, no report is published on therapeutic approach for diseases, which approach comprises the inhibition of the signal transduction of TGF-β mediated by TGF-β type II receptor using antibodies against the TGF-β type II receptor.
Polyclonal antibodies derived from non-human mammals, such as rabbit and goat, are the only antibodies reported as antibodies against human TGF-β type II receptor. So far, the preparation of human-derived monoclonal antibodies and attempts of therapy for various diseases using human monoclonal antibody, not to mention monoclonal antibodies against human TGF-β type II receptor have been reported.